Drawing on convergent mixed methods research design, this study examined Junior High School (JHS) mathematics teachers’ practice of differentiated instruction and its associated challenges in Tano South district, in the Ahafo Region of Ghana. Simple random sampling technique was used to select 50 JHS mathematics teachers for the quantitative study with the aid of questionnaire, purposive sampling technique was used to solicit qualitative data for the study using interview guide. Inferential statistics and thematic analysis were used to analysis the quantitative and qualitative data respectively. It was generally observed that, there was no significant differences between special and general education teachers practice of differentiated instruction. Therefore, the study recommends that Ghana Education Service (GES) and stakeholders in education should implement professional development and training programmes for both general and special mathematics teachers on differentiated instruction.